Genetic structure of drone congregations of the stingless bee Scaptotrigona mexicana

Drones of stingless bee species often form distinctive congregations of up to several hundred individuals which can persist over considerable periods of time. Here we analyse the genetic structure of three drone congregations of the neotropical stingless bee Scaptotrigona mexicana employing eight microsatellite markers. Two congregations were close to each other (50 m), the third one was located more than 10 km away from them. This spatial pattern was also reflected on the genetic level : the two close congregations did not show any population sub-structuring, whereas the more distant congregation showed a significant population differentiation to both of them. Population subdifferentiation was however low with F _st values (F _st = 0.020 and 0.014) between the distant congregations, suggesting gene flow over larger distances mediated by the drones of S. mexicana. Based on the genotypic data we also estimated the number of colonies contributing drones to the congregations. The two joint congregations consisted of drones originating from 39,6 colonies, while the third congregation was composed of drones from 21,8 colonies, thus proving that congregations of S. mexicana are constituted of unrelated drones of multicolonial origin. Received 23 April 2007; revised 21 September 2007; accepted 2 October 2007.     

Genetic structure of nest aggregations and drone congregations of the southeast Asian stingless bee Trigona collina

In stingless bees, sex is determined by a single complementary sex-determining locus. This method of sex determination imposes a severe cost of inbreeding because an egg fertilized by sperm carrying the same sex allele as the egg results in a sterile diploid male. To explore how reproductive strategies may be used to avoid inbreeding in stingless bees, we studied the genetic structure of a population of 27 colonies and three drone congregations of Trigona collina in Chanthaburi, Thailand. The colonies were distributed across six nest aggregations, each aggregation located in the base of a different fig tree. Genetic analysis at eight microsatellite loci showed that colonies within aggregations were not related. Samples taken from three drone congregations showed that the males were drawn from a large number of colonies (estimated to be 132 different colonies in our largest swarm). No drone had a genotype indicating that it could have originated from the colony that it was directly outside. Combined, these results suggest that movements of drones and possibly movements of reproductive swarms among colony aggregations provide two mechanisms of inbreeding avoidance.     

A Game Theoretical Analysis of the Mating Sign Behavior in the Honey Bee

Queens of the honey bee, Apis mellifera (L.), exhibit extreme polyandry, mating with up to 45 different males (drones). This increases the genetic diversity of their colonies, and consequently their fitness. After copulation, drones leave a mating sign in the genital opening of the queen which has been shown to promote additional mating of the queen. On one hand, this signing behavior is beneficial for the drone because it increases the genetic diversity of the resulting colony that is to perpetuate his genes. On the other hand, it decreases the proportion of the drone??s personal offspring among colony members which is reducing drone fitness. We analyze the adaptiveness and evolutionary stability of this drone??s behavior with a game-theoretical model. We find that theoretically both the strategy of leaving a mating sign and the strategy of not leaving a mating sign can be evolutionary stable, depending on natural parameters. However, the signing strategy is not favored for most scenarios, including the cases that are biologically plausible in reference to empirical data. We conclude that leaving a sign is not in the interest of the drone unless it serves biological functions other than increasing subsequent queen mating chances. Nevertheless, our analysis can also explain the prevalence of such a behavior of honey bee drones by a very low evolutionary pressure for an invasion of the nonsigning strategy.     

Drone and Worker Brood Microclimates Are Regulated Differentially in Honey Bees,Apis mellifera

Background

Honey bee (Apis mellifera) drones and workers show differences in morphology, physiology, and behavior. Because the functions of drones are more related to colony reproduction, and those of workers relate to both survival and reproduction, we hypothesize that the microclimate for worker brood is more precisely regulated than that of drone brood.

Methodology/Principal Findings

We assessed temperature and relative humidity (RH) inside honey bee colonies for both drone and worker brood throughout the three-stage development period, using digital HOBO^® Data Loggers. The major findings of this study are that 1) both drone and worker castes show the highest temperature for eggs, followed by larvae and then pupae; 2) temperature in drones are maintained at higher precision (smaller variance) in drone eggs and larvae, but at a lower precision in pupae than the corresponding stages of workers; 3) RH regulation showed higher variance in drone than workers across all brood stages; and 4) RH regulation seems largely due to regulation by workers, as the contribution from empty honey combs are much smaller compared to that from adult workers.

Conclusions/Significance

We conclude that honey bee colonies maintain both temperature and humidity actively; that the microclimate for sealed drone brood is less precisely regulated than worker brood; and that combs with honey contribute very little to the increase of RH in honey bee colonies. These findings increase our understanding of microclimate regulation in honey bees and may have implications for beekeeping practices.     

Extensive population admixture on drone congregation areas of the giant honeybee,Apis dorsata (Fabricius, 1793)

The giant honeybee Apis dorsata often forms dense colony aggregations which can include up to 200 often closely related nests in the same location, setting the stage for inbred matings. Yet, like in all other Apis species, A. dorsata queens mate in mid‐air on lek like drone congregation areas (DCAs) where large numbers of males gather in flight. We here report how the drone composition of A. dorsata DCAs facilitates outbreeding, taking into the account both spatial (three DCAs) and temporal (subsequent sampling days) dynamics. We compared the drones’ genotypes at ten microsatellite DNA markers with those of the queen genotypes of six drone‐producing colonies located close to the DCAs (Tenom, Sabah, Malaysia). None of 430 sampled drones originated from any of these nearby colonies. Moreover, we estimated that 141 unidentified colonies were contributing to the three DCAs. Most of these colonies were participating multiple times in the different locations and/or during the consecutive days of sampling. The drones sampled in the DCAs could be attributed to six subpopulations. These were all admixed in all DCA samples, increasing the effective population size an order of magnitude and preventing matings between potentially related queens and drones.     

Temporal variation in the genetic structure of a drone congregation area: an insight into the population dynamics of wild African honeybees (Apis mellifera scutellata)

The mating system of the honeybee ( Apis mellifera ) has been regarded as one of the most panmictic in the animal kingdom, with thousands of males aggregating in drone congregation areas (DCAs) that virgin queens visit to mate with tens of partners. Although males from many colonies gather at such congregations, the temporal changes in the colonies contributing drones remain unknown. Yet, changes in the DCAs' genetic structure will ultimately determine population gene flow and effective population size. By repeatedly sampling drones from an African DCA over a period of 3 years, we studied the temporal changes in the genetic structure of a wild honeybee population. Using three sets of tightly linked microsatellite markers, we were able to reconstruct individual queen genotypes with a high accuracy, follow them through time and estimate their rate of replacement. The number of queens contributing drones to the DCA varied from 12 to 72 and was correlated with temperature and rainfall. We found that more than 80% of these queens were replaced by mostly unrelated ones in successive eight months sampling intervals, which resulted in a clear temporal genetic differentiation of the DCA. Our results suggest that the frequent long-range migration of colonies without nest-site fidelity is the main driver of this high queen turnover. DCAs of African honeybees should thus be regarded as extremely dynamic systems which together with migration boost the effective population size and maintain a high genetic diversity in the population.     

Investigation of polyandry in honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>) using microsatellites

Polyandry is a specific phenomenon increasing genetic diversity. To analyze the level of polyandry and to assess the contribution of drones to the genetic diversity of honey bee colonies, three microsatellite loci (A008, Ap049, and AC117) were studied in honey bees from colonies of different origin (Middle Russian race, Carpathian race, and hybrids) in Tomsk Province. The share of the introduced paternal alleles varied between colonies from 6.67 to 28.00%. The highest genetic diversity was observed in hybrid colonies (25–28% of introduced paternal alleles).     

Assessing the density of honey bee colonies at ecosystem scales

1. Information about the density of wild honey bee (Apis spp.) colonies in an ecosystem is central to understanding the functional role of honey bees in that ecosystem, necessary for effective biosecurity response planning, and useful for determining whether pollination services are adequate. However, direct visual surveys of colony locations are not practical at ecosystem scales. Thus, indirect methods based on population genetic analysis of trapped males have been proposed and implemented. 2. In this review, indirect methods of assessment of honey bee colony densities are described, which can be applied at ecosystem scales. The review also describes how to trap males in the field using the Williams drone trap (or virgin queens) the appropriate genetic markers and statistical analyses, and discusses issues surrounding sample size. 3. The review also discusses some outstanding issues concerning the methods and the conversion of estimated colony number to colony density per km². The appropriate conversion factor will require further research to determine the area over which a drone trap draws drones.     

Relatedness among honeybees (Apis mellifera) of a drone congregation

The honeybee (Apis mellifera) queen mates during nuptial flights, in the so-called drone congregation area where many males from surrounding colonies gather. Using 20 highly polymorphic microsatellite loci, we studied a sample of 142 drones captured in a congregation close to Oberursel (Germany). A parentage test based on lod score showed that this sample contained one group of four brothers, six groups of three brothers, 20 groups of two brothers and 80 singletons. These values are very close to a Poisson distribution. Therefore, colonies were apparently equally represented in the drone congregation, and calculations showed that the congregation comprised males that originated from about 240 different colonies. This figure is surprisingly high. Considering the density of colonies around the congregation area and the average flight range of males, it suggests that most colonies within the recruitment perimeter delegated drones to the congregation with an equal probability, resulting in an almost perfect panmixis. Consequently, the relatedness between a queen and her mates, and hence the inbreeding coefficient of the progeny, should be minimized. The relatedness among the drones mated to the same queen is also very low, maximizing the genetic diversity among the different patrilines of a colony.     

Host preference of the honey bee tracheal mite (Acarapis woodi (Rennie))

Field and laboratory bioassays were used to test the preference of the honey bee tracheal mite,Acarapis woodi (Rennie), for drones versus workers. Groups of newly-emerged drones and workers were marked and introduced into either heavily infested colonies (field bioassays) or into the cages of infested bees obtained from the field colonies (laboratory bioassays). Seven days later all of the marked bees in each bioassay were removed. The numbers of mites of each life stage in each drone or worker target bee of each experiment were quantified. Mite prevalence values for the two castes were not found to differ significantly for either experiment. However, the caste of the target bee was shown to influence the migration of the adult female mites. Drones contained a greater number of migratory female mites and greater total numbers of all mite stages as compared to workers. These results indicate that migrating female mites preferentially infest drones and suggest that the role of drones in the dissemination and population dynamics of the tracheal mite needs to be examined further.     

What's in that package? An evaluation of quality of package honey bee (Hymenoptera: Apidae) shipments in the United States

To replace deceased colonies or to increase the colony numbers, beekeepers often purchase honey bees, Apis mellifera L., in a package, which is composed of 909-1,364 g (2-3 lb) of worker bees and a mated queen. Packages are typically produced in warm regions of the United States in spring and shipped throughout the United States to replace colonies that perished during winter. Although the package bee industry is effective in replacing colonies lost in winter, packages also can be an effective means of dispersing diseases, parasites, and undesirable stock to beekeepers throughout the United States. To evaluate the quality of packages, we examined 48 packages representing six lines of bees purchased in the spring 2006. We estimated levels of the parasitic mite Varroa destructor Anderson & Trueman and the percentage of drone (male) honey bees received in packages. We surveyed for presence of the tracheal honey bee mite, Acarapis woodi (Rennie), and a microsporidian parasite, Nosema spp., in the shipped bees. We found significant differences in both the mean Varroa mite per bee ratios (0.004-0.054) and the average percentage of drones (0.04-5.1%) in packages from different producers. We found significant differences in the number of Nosema-infected packages (0.0-75.0%) among the six lines. No packages contained detectable levels ofA. woodi. Considering the observed variability among honey bee packages, beekeepers should be aware of the potential for pest and disease infestations and high drone levels in packages.     

The effect of induced changes in sexual asymmetry of honey bees (Apis mellifera) on swarming behaviour

This study was built on the assumption that mother (queen) and workers (nurses) distribute their genes either through swarms (female biomass) or through the drones (male biomass). The swarming mood of the bee colonies was suppressed by an exactly defined increase in drone rearing. We studied the efficiency of reproductive investments (on genetic and energetic levels) of the mother and workers to the next generations. The equalization of fitness of the mother and nurses was achieved by a deliberately induced change in numerically stable sex asymmetry of a bee colony. A swarm was compensated with its energy demand and a volume ratio of distributed genetic information. The newly introduced term “reproductive investment complex” (RIC) includes the reproductive potential of the mother and reproductive energy of workers into care for the mother and for the brood. The number of individuals of one sex was closely connected with the weight of individuals of the oppposite sex. The described method of suppression of swarming mood was successfully tested on 60 honey bee colonies over seven years (2003–2009). A number of beekeepers that were acquainted with this method confirmed the success.     

Drone “quality” and caste interactions in the honey bee, Apis mellifera L.

We investigated the influence of drone size and potential reproductive quality on caste interactions by adding large drones reared in drone cells (DC drones; considered to be of higher quality) and small drones reared in worker cells (WC drones; of lower quality) to two observation colonies and monitoring worker–drone interactions and acceptance by workers. When initially introduced into the colonies more DC drones received trophallaxis, whereas more WC drones received aggression and eviction attempts from workers. Nevertheless, WC and DC drones were equally likely to be accepted by workers. For both drone types accepted individuals had slightly, but significantly greater weights than rejected males. Thus, workers discriminated between drones of different sizes and potential quality upon initial encounter, although these discriminations were not strongly associated with acceptance decisions. After drones were accepted, workers either showed no preference for interacting with WC or DC drones, or if a preference was shown it tended to favor WC drones. Compared to accepted DC drones, significantly more WC drones received grooming for longer periods of time and also spent more time engaged in all interactions with workers combined. DC and WC drones did not differ in the likelihood of receiving trophallaxis or the vibration signal, although for both interactions slightly more WC drones were recipients. Thus, workers may bias some interactions with accepted drones to favor smaller individuals with potential developmental deficiencies, in a manner that could contribute to the production of a greater total number of competitive males and increased colony reproductive output.     

Determining colony densities in wild honeybee populations (<Emphasis Type="Italic">Apis mellifera</Emphasis>) with linked microsatellite DNA markers

Estimating the population size of social bee colonies in the wild is often difficult because nests are highly cryptic. Because of the honeybee (Apis mellifera) mating behaviour, which is characterized by multiple mating of queens at drone congregation areas (DCA), it is possible to use genotypes of drones caught at these areas to infer the number of colonies in a given region. However, DCAs are difficult to locate and we assess the effectiveness of an alternative sampling technique to determine colony density based on inferring male genotypes from queen offspring. We compare these methods in the same population of wild honeybees, Apis mellifera scutellata. A set of linked microsatellite loci is used to decrease the frequency of recombination among marker loci and therefore increase the precision of the estimates. Estimates of population size obtained through sampling of queen offspring is significantly larger than that obtained by sampling drones at DCAs. This difference may be due to the more extensive flying range of queens compared with drones on mating flights. We estimate that the population size sampled through queen offspring is about double that sampled through drones.     

Three QTL in the honey bee Apis mellifera L. suppress reproduction of the parasitic mite Varroa destructor

Varroa destructor is a highly virulent ectoparasitic mite of the honey bee Apis mellifera and a major cause of colony losses for global apiculture. Typically, chemical treatment is essential to control the parasite population in the honey bee colony. Nevertheless a few honey bee populations survive mite infestation without any treatment. We used one such Varroa mite tolerant honey bee lineage from the island of Gotland, Sweden, to identify quantitative trait loci (QTL) controlling reduced mite reproduction. We crossed a queen from this tolerant population with drones from susceptible colonies to rear hybrid queens. Two hybrid queens were used to produce a mapping population of haploid drones. We discriminated drone pupae with and without mite reproduction, and screened the genome for potential QTL using a total of 216 heterozygous microsatellite markers in a bulk segregant analysis. Subsequently, we fine mapped three candidate target regions on chromosomes 4, 7, and 9. Although the individual effect of these three QTL was found to be relatively small, the set of all three had significant impact on suppression of V. destructor reproduction by epistasis. Although it is in principle possible to use these loci for marker-assisted selection, the strong epistatic effects between the three loci complicate selective breeding programs with the Gotland Varroa tolerant honey bee stock.     

The honeybee queen influences the regulation of colony drone production

Social insect colonies invest in reproduction and growth, buthow colonies achieve an adaptive allocation to these life-historycharacters remains an open question in social insect biology.Attempts to understand how a colony's investment in reproductionis shaped by the queen and the workers have proved complicatedbecause of the potential for queen–worker conflict overthe colony's investment in males versus females. Honeybees,in which this conflict is expected to be minimal or absent,provide an opportunity to more clearly study how the actionsand interactions of individuals influence the colony's productionand regulation of males (drones). We examined whether honeybeequeens can influence drone regulation by either allowing orpreventing them from laying drone eggs for a period of timeand then examining their subsequent tendency to lay drone andworker eggs. Queens who initially laid drone eggs subsequentlylaid fewer drone eggs than the queens who were initially preventedfrom producing drone eggs. This indicates that a colony's regulationof drones may be achieved not only by the workers, who buildwax cells for drones and feed the larvae, but also by the queen,who can modify her production of drone eggs. In order to betterunderstand how the queen and workers contribute to social insectcolony decisions, future work should attempt to distinguishbetween actions that reflect conflict over sex allocation andthose that reflect cooperation and shared control over the colony'sinvestment in reproduction.     

Successful maintenance of a stingless bee population despite a severe genetic bottleneck

Stingless bees play an important ecological role as pollinators of many wild plant species in the tropics and have significant potential for the pollination of agricultural crops. Nevertheless, conservation efforts as well as commercial breeding programmes require better guidelines on the amount of genetic variation that is needed to maintain viable populations. In this context, we carried out a long-term genetic study on the stingless bee Melipona scutellaris to evaluate the population viability consequences of prolonged breeding from a small number of founder colonies. In particular, it was artificially imposed a genetic bottleneck by setting up a population starting from only two founder colonies, and continued breeding from it for a period of over 10?years in a location outside its natural area of occurrence. We show that despite a great reduction in the number of alleles present at both neutral microsatellite loci and the sex-determining locus relative to its natural source population, and an increased frequency in the production of sterile diploid males, the genetically impoverished population could be successfully bred and maintained for at least 10?years. This shows that in stingless bees, breeding from a small stock of colonies may have less severe consequences than previously suspected. In addition, we provide a simulation model to determine the number of colonies that are needed to maintain a certain number of sex alleles in a population, thereby providing useful guidelines for stingless bee breeding and conservation efforts.     

Genetic structure of drone congregation areas of Africanized honeybees in southern Brazil

As yet, certain aspects of the Africanization process are not well understood, for example, the reproductive behavior of African and European honeybees and how the first Africanized swarms were formed and spread. Drone congregation areas (DCAs) are the ideal place to study honeybee reproduction under natural conditions since hundreds of drones from various colonies gather together in the same geographical area for mating. In the present study, we assessed the genetic structure of seven drone congregations and four commercial European-derived and Africanized apiaries in southern Brazil, employing seven microsatellite loci for this purpose. We also estimated the number of mother-colonies that drones of a specific DCA originated from. Pairwise comparison failed to reveal any population sub-structuring among the DCAs, thus indicating low mutual genetic differentiation. We also observed high genetic similarity between colonies of commercial apiaries and DCAs, besides a slight contribution from a European-derived apiary to a DCA formed nearby. Africanized DCAs seem to have a somewhat different genetic structure when compared to the European.     

Linkage analysis of sex determination in the honey bee (Apis mellifera)

A colony-level phenotype was used to map the major sex determination locus (designatedX) in the honey bee (Apis mellifera). Individual queen bees (reproductive females) were mated to single drones (fertile males) by instrumental insemination. Haploid drone progeny of an F1 queen were each backcrossed to daughter queens from one of the parental lines. Ninety-eight of the resulting colonies containing backcross progeny were evaluated for the trait ‘low brood-viability’ resulting from the production of diploid drones that were homozygous atX. DNA samples from the haploid drone fathers of these colonies were used individually in polymerase chain reactions (PCR) with 10-base primers. These reactions generated random amplified polymorphic DNA (RAPD) markers that were analyzed for cosegregation with the colony-level phenotype. One RAPD marker allele was shared by 22 of 25 drones that fathered low brood-viability colonies. The RAPD marker fragment was cloned and partially sequenced. Two primers were designed that define a sequence-tagged site (STS) for this locus. The primers amplified DNA marker fragments that cosegregated with the original RAPD marker. In order to more precisely estimate the linkage betweenX and the STS locus, another group of bees consisting of progeny from one of the low-brood viability colonies was used in segregation analysis. Four diploid drones and 181 of their diploid sisters (workers, nonfertile females) were tested for segregation of the RAPD and STS markers. The cosegregating RAPD and STS markers were codominant due to the occurrence of fragment-length alleles. The four diploid drones were homozygous for these markers but only three of the 181 workers were homozygotes (recombinants). Therefore the distance betweenX and the STS locus was estimated at 1.6 cM. An additional linked marker was found that was 6.6 cM from the STS locus.